Multi-drug resistant (MDR) bacterial infections represent one of the most serious challenges facing health care today. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of hospital-acquired infections, and is particularly problematic for patients who are immunosuppressed or require mechanical ventilation. P. aeruginosa persists chronically in cystic fibrosis patients, resulting in irreversible lung damage and mortality. Both natural and acquired characteristics, including the expression of several multidrug efflux systems, make P. aeruginosa infections especially difficult to treat. In addition to its intrinsic drug resistance, the infectivity of P. aeruginosa is enhanced by expression of a Type III secretion system (T3SS). This needle-type apparatus directly injects effector proteins into eukaryotic host cells to facilitate bacterial surival and dissemination. The most cytotoxic T3SS effector produced by P. aeruginosa is the phospholipase, ExoU. This application builds upon our previous discovery that ubiquitin and ubiquitinated proteins act as essential cofactors for the activation of ExoU. The interaction of ExoU with ubiquitin is novel in that it does not involve the covalent attachment or removal of ubiquitin subunits, leading us to hypothesize that ubiquitin acts as a scaffold to induce the folding of ExoU into a catalytically-active conformation. We will employ innovative site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) methods in conjunction with genetic and biochemical approaches to characterize the structure of ExoU in solution, elucidate the structure of the ExoU-ubiquitin complex, and investigate ExoU-membrane interactions. These studies will advance the application of new methods to investigate protein-protein interactions, facilitate the development of novel inhibitors of ExoU virulence, and promote our understanding of bacterial toxins that target the membrane interface.